It is proposed to develop in vitro assays to measure antigen specific proliferative and cytotoxic responses in acute and chronic hepatitis B virus (HBV) infection. In the absence of adequate cell culture for propagation of HBV, the major problem has been the lack of an appropriate stimulator/target cell. Autologous, peripheral blood mononuclear cells (PBMC) chemically coupled with purified hepatitis B surface antigen (HBsAg) will serve as stimulator cells in proliferative and cytotoxic assays and also serve as target cells in the cytotoxic assays. A modified CrCl3 procedure has been evaluated for efficiency of coupling HBsAg and toxicity to PBMC and the results appear encouraging. Alternatively, a method utilizing a bisdiazotized benzidine reagent will be tested. Autologous responder and stimulator cells will be co-cultured for proliferation and cytotoxicity against autologous target cells. Since circulating cytotoxic T-lymphocytes (CTL) may be present in peripheral blood only in acute HBV infection, this in vitro sensitization process will allow for the generation of CTL in chronically infected patients. Similarly, the use of autologous and allogeneic stimulator and target cells will provide a system in which the influence of HLA antigens on proliferative and cytotoxic responses can be assessed. In addition, development of such assays will allow analysis of the subpopulations involved in HBsAg-specific proliferation and cytotoxicity as well as facilitate the demonstration of suppressive factors in serum and/or culture supernatants of chronic HBV patients.